Insulin resistance is a critical factor in the pathogenesis of Type 2 Diabetes and cardiovascular diseases, and is due to impaired stimulation of glucose uptake in skeletal muscle. Insulin resistance is also associated with abnormalities in fat metabolism that exist independent of the degree of generalized obesity, including elevated circulating free fatty acids and increased accumulation of intramuscular lipid. Our central hypothesis is that the accumulation of intramyocellular lipid (IML) involves defects muscle fat metabolism, and is integrally related to impaired function of the glucose transport system. Our goal is for the first time to elucidate the molecular basis of abnormal muscle lipid metabolism in insulin resistant humans. Study groups include both lean and obese insulin-sensitive and - resistant individuals to discriminate between effects of obesity and insulin resistance, treatment with troglitazone and dexamethasone, and Type 2 diabetics before and after intensive euglycemic therapy. In specific aim 1, we will examine the relationship between insulin resistance and increased intramuscular lipid, and whether lipid accumulation is due to a relative defect in oxidation and/or an increase in FFA delivery. These studies will employ hyperinsulinemic clamps, palmitate turnover, AV leg balance studies, and whole-body and leg indirect calorimetry. IML will be assessed by histology and proton NMR spectroscopy. In specific aim 2, carnitine palmitoyltransferase 1 (CPT1), malonyl CoA, and acetyl CoA carboxylase will be examined in skeletal muscle as critical determinants of fuel partitioning and insulin sensitivity. In specific aim 3, in situ microdialysis will be used to study the role of TNFalpha as an autocrine/paracrine factor in regulating fatty acid metabolism and insulin sensitivity in adipose and muscle tissues The hypotheses are that dysregulation of this fuel sensing apparatus will reduce muscle CPT1 activity, inhibit entry and oxidation of long chain acyl CoAs in mitochondria, and promote accumulation of IML. Specific aim 4 will assess structure/function relationships for novel common polymorphisms in the muscle CPT1 gene that are associated with hyperglycemia, hyperinsulinemia, and insulin resistance. Fat metabolism, IML, CPT1 activity, and insulin sensitivity will be examined in vivo as a function of genotype, and specific activity of CPT1 variants will be assessed expressed in yeast. These experiments will for the first time examine relationships between muscle fat metabolism, IML, TNFalpha and insulin sensitivity in humans.